P443: A POOLED FOCUSED IN VIVO CRISPR-CAS9 SCREENING IN IMMUNOCOMPETENT MICE ALLOWS IDENTIFICATION OF IMMUNE CHECKPOINTS RELEVANT FOR LEUKEMIC STEM CELLS
Authors: Chantal Bachmann, Stefan Forster, Adrian Ochsenbein
Journal: HemaSphere (2023)

Background: Acute myeloid leukaemia (AML) is initiated and maintained by leukemic stem cells (LSCs). Due to their increased resistance to conventional therapies, they are responsible for disease relapse. Therefore, specific strategies targeting and eliminating LSCs are needed. Cells of the tumour microenvironment, including stromal cells, blood vessels and immune cells, are involved in the regulation of LSCs and their stem cell properties. Immune checkpoint receptors and ligands are well known for their role in fine-tuning the immune response, but their expression has also been described on cancer and leukemic stem cells, where they can play important roles in the regulation of stem cell function. Aims: Our aim was to develop a pooled, focused in vivo CRISPR-Cas9 screening to study the role of immune checkpoint molecules in the regulation of LSCs. Importantly, in contrast to existing screenings, we aimed to minimize introduction of artefacts and disruption of the tumour microenvironment by avoiding irradiation of recipient mice. Methods: AML was induced by transplanting retrovirally MLL-AF9 transduced Cas9-expressing LSKs into primary recipient mice. LSCs were isolated from leukemic mice and the lentiviral sgRNA library was introduced into the LSCs at a low MOI. Library+ cells were enriched and used for the screening in stem cell assays such as methylcellulose colony formation assay or secondary leukaemia development. Validation of effects was performed by using CRISPR RNP on LSCs. Results: In vitro culture conditions and handling procedures for primary murine AML LSCs were optimized so that cells would retain stemness and survive cell stress induced by ex vivo culture, fluorescence-activated cell sorting (FACS), library transduction and enrichment. mCherry and Cas9 proteins resulted in rejection of LSCs in secondary recipient mice, thus requiring adaption of the recipient mice, sgRNA library and disease monitoring strategy. Finally, a screening targeting 25 immune checkpoint molecules, three positive enrichment controls and three positive dropout controls was performed in vitro, in sublethally irradiated syngeneic recipient mice, in RAG1 KO mice, and in non-irradiated syngeneic recipient mice. In the in vivo screening, we successfully retrieved a positive dropout control gene and a positive enrichment control gene, confirming the technical feasibility of the screening. Additionally, possible effects of gene knockouts on LSCs transplanted into irradiated recipient mice seemed to be lost in the non-irradiated recipients. This confirms the importance of non-irradiated recipient mice for an artefact-free screening of genes hypothesized to be involved in interactions of LSCs and the tumour microenvironment. Summary/Conclusion: We have established a pooled, focused in vivo CRISPR-Cas9 screening in a murine model of MLL-AF9-driven AML that allows the artefact-free discovery of genes important for LSCs and their interactions with the tumour microenvironment. Flexible library design allows to quickly study a set of different genes, while the utilisation of different stem cell assays or secondary recipient mice helps in estimating contributions from artefacts or different tumour microenvironment components.Keywords: Stem cell niche, Microenvironment, Leukemic stem cell, Acute myeloid leukemia